KR200307065Y1 - Synthesizer for Molecular Diversity and Apparatus for Purification - Google Patents

Abstract

The present invention relates to a synthesis apparatus related to the construction of new drug candidates in the field of new drug development, a standardized equipment that can purify synthesized compounds with bioactive screening quality, and an experimental system. It is possible to purify several compounds at the same time with the collectively constructed compound.

Description

Synthesizer for Molecular Diversity and Apparatus for Purification

The present invention is a standard for collectively constructing new drug candidate compounds as a technology related to compound diversity, which takes the most time in the search for tens of thousands of new drug candidates and new drug candidates as the genome business is nearing completion. The present invention relates to the development of a standard multi-purification device capable of collectively purifying several compounds simultaneously in purifying a collective reaction device and collectively synthesized compounds. The purpose of the present invention is to provide a compound library with a quality that can satisfy the quality for activity detection of a compound prepared by a combinatorial method in a compound preparation management-based technology in a short time.

As the genome business grows, life sciences begin to transform functional information from the genome into protein information, understand their role, and regulate their function, or functional genomics. Information from functional genomics is expected to fully interpret the function and function of living organisms, enabling the treatment of most diseases facing humanity.

Completion of the genome project will enable the development of more than tens of thousands of new drug development tasks, simply hanging on to hundreds of new disease treatment development tasks. Therefore, new drug candidate compound construction technology is inevitably required in order to link the vast amount of information obtained from the era of genome business completion to the research and development of practical new drugs. This will require the cooperation of several scientific disciplines, including biology, mathematics, computational science, and chemistry, to solve the problem of developing tens of thousands of new drugs in the shorter period of new drug development. The efforts to build various compounds have been made in various fields such as the development of an automatic compound synthesizer and the development of an automatic robot for ultrafast compound activity retrieval.

Looking at the existing drug development process, even after the cause of the disease is identified, it takes a long time to develop a therapeutic agent, and in particular, a large number of compounds are needed to derive candidate substances that can be therapeutic agents. The development of therapeutics in the last 20th century suggests that only one out of approximately 10,000 compounds is available as a therapeutic. Now, if functional targets are increased to tens of thousands through functional genomics, more than hundreds of millions of compounds are needed right now to develop therapeutics. In this situation, it is important to rapidly and efficiently synthesize drug-related candidate compounds. These compounds will play a more important role in the search for disease-related targets by varying the research methods and concepts of finding candidates from existing target identification.

Against this background, recent drug development trends are focused on technologies for synthesizing tens of thousands of compounds simultaneously in a method called combinatorial chemistry to detect biological activity at once. This is the most efficient technique for finding new drug candidates and finding new drug candidates with high activity in the lead derivatives. Combination chemistry has been used to construct numerous compounds based on peptide synthesis since the 1960s, and until recently, millions of compounds have been synthesized by constructing compounds using solid phase supports of combinatorial chemistry. However, the existing compound is constructed with a focus on the number of compounds within a short time. All combinatorial chemical synthesis is performed without purification of a substance that can act as a false positive in the actual compound activity search. lost.

In the 1990s, combinatorial devices developed in the form of automated synthesis systems did not show the function of well-refined compounds according to small-scale traditional synthesis in the introduction of compound activity screening systems. In other words, when a compound is synthesized chemically, a polystyrene-based polymer compound, which is used as a solid support, is separated from the compound at the end of the synthesis by UV spectrometer, NMR, IR, MS spectrometer. As included in the present invention, it was accompanied by a remarkable experimental difference in the retrieval activity with a compound that is mostly purified by silica gel column in the conventional synthetic method.

Due to these experimental errors, recent compound construction has been shifted to the synthesis and construction of compounds in the form of at least filtration. In other words, who can synthesize and purify a large number of compounds in the fastest way to build a compound best understands the compound construction necessary for drug development.

Combination chemical reaction equipment and devices have already been well developed by numerous companies and are now used by many chemists to modify the equipment and devices on the market with the use of the equipment or to be calibrated to meet the purpose of use. It has been. However, in most compound construction systems, there is no application filed as a whole system other than those characteristic of one step. In addition, since the development including the synthesis after the purification is not carried out at all, it is necessary to be purified from the synthesis or the existing mixture again in order to search for the correct activity for the compounds that have been synthesized and completed the compound activity search.

Therefore, it is important to know how fast a compound can be synthesized now, but how fast it is possible to simultaneously purify a large number of compounds in a short time has been of great interest in compound construction. Therefore, a new type of synthesis equipment needs to be developed to satisfy this.

Therefore, the present invention intends to develop new equipment that can be a standard in building a compound by complementing the shortcomings of these.

The invention is largely divided into three configurations, firstly, a novel reactor sealing frame for solving the problem of leakage of a credit medium and a reactant capable of synthesizing a compound, and secondly a reactant in a reactor equipped with a reactor and a sealing frame. It is a rotary mixing device that mixes. Third is a purification system capable of simultaneously purifying eight synthesized compounds collectively. It is composed of eight purification columns consisting of a device for solvent storage and dispensing and a set. In particular, the sealing frame is formed by connecting rods to the upper and lower plates (Figs. 2 to 4) and the intermediate frame to completely connect the upper and lower plates (Figs. 5 to 9). To two) forms.

The present invention employs a new type of rod-and-tightening frame that can completely block the leakage of solvent and reactant liquid, which is a disadvantage of the existing commercialized synthetic equipment in constructing a compound library in a solid phase combinatorial chemistry. It is manufactured by using a gasket made of an inert material under acid and base conditions, and by introducing a simultaneous refining device and installing a 360-degree rotating stirring device to completely eliminate the inconvenience of solvent leakage due to natural solution leakage or excessive stirring of the existing device. Excellent effect that can be dissolved and react safely and conveniently, the synthetic compounds can be separated and purified at the same time instant reaction to obtain a pure form of the compound, the activity test, and the strong sealing effect to maximize the stirring power Can be rotated 360 degrees of stirring device for this purpose It is possible to change to the form of, so that it is possible to increase the reaction rate by increasing the stirring efficiency by escaping the stirring form of the vertical stirring form of the existing stirring type, the effective reaction of the reactants in the reactor by the 360 degree rotary stirrer The reaction liquid moves along the inner end of the reactor to reduce the damage to the solid support by the existing vortex or stirrer type. Overcomes the phenomenon of slack and plays a role in promoting the reaction.

As described above, the technical problem to be achieved in the present invention is applied to the basic technology for standardizing a common experimental procedure in constructing a compound library chemically. The compound library is the fastest and most economical method for compound synthesis using a solid phase support. It is to build a compound library that can be prepared and the purification of the prepared compound is the fastest, and meets the requirements of the quality of bioactive screening that must be performed as a drug candidate. In addition, the rotary mixer for reaction mixing can reduce the damage of the solid support by the conventional vortex or stirrer type by moving the reactant liquid along both ends of the reactor to allow the effective reaction of the reactants in the reactor. In addition, the articulators by vibrating type of the entire reactor play a role of promoting the reaction by overcoming the phenomenon of loosening during the reaction.

Hereinafter, the present invention will be described in detail.

The reactor, which is used as part of the synthesis apparatus and equipment of the present invention, has 96-well and 48-well, each having the same shape as that of FIG. Below the well (1) is a reactor for the preparation of a compound library using a solid phase support equipped with a filtration frit 3 '. As shown in FIG. 1, the well 1 represents a reaction well capable of chemical reaction of about 5.0 ml, and the partition 2 represents a function of separating each well from the well. (1) is the outlet 3 of the lower part where the filtration frit 3 'is fixed so that the solvent or the solute can be moved after the chemical reaction.

The sealing frame of the present invention necessary for constructing the synthesizing apparatus and equipment of the present invention has a configuration that can prevent the leakage of the solvent in the present invention is composed of two forms in the present invention.

The first is a through frame with rods as shown in the top view of FIG. That is, as shown in Figs. 2 to 4, the rods 4 and 9 are provided on the upper plate 5 and the lower plate 8 of the frame, and the holes of the upper plate and lower plate gasket parts 6 and 10 are provided. (7, 11) is a frame in which the upper and lower rods, which are formed by tightening the upper and lower plates with bolts 12 and 13, through the holes of the gasket part of the upper and lower plates. to be. By having such a configuration, in a multistage reaction, that is, when a two-stage reaction is performed by adding a new reactant after the one-stage reaction, only the joint part connecting the lower plate and the upper plate is removed and the rest is not removed. Provides a frame capable of performing the reaction quantitatively and accurately without leakage of the solution by the outlet 3 below 1a (a))

Second is the flip frame shown in the top view of FIG. That is, as shown in FIGS. 5 to 9, a frame composed of a lower plate 17, an upper plate frame 21 and a lower plate frame 22, and an upper plate 15, which assist in the sealing role and effective mixing of the reactor by the frame. The lower plate 17 and the lower plate frame 22, the upper plate 15 and the upper plate frame 21 are connected to each other by flip joints by flip joints 23. 23 '. Since the connection and detachment is a screw type, it is possible to facilitate the function to control the adhesion force through the strength of the connection bolt of the joint. With this configuration, in the multi-step reaction, that is, when adding a new reactant after the one-step reaction, and performing the two-step reaction, only the bolt part connected to the upper frame and the upper plate, the lower plate and the lower plate frame is removed, and the rest is not detached. This provides a frame in which the reaction can be carried out quantitatively and accurately without leakage of the solution by the outlet 3 under the reactor (FIG. 1 (a)).

Looking at each of the drawings showing the coupling relationship of these devices are as follows. FIG. 2 is a view showing a state in which the frame is dismantled without combining a 48-well or 96-well reactor (FIG. 1 (a)) in each configuration constituting the penetrating frame. (4) denotes a rod provided on the upper plate of the upper rod part, (5) is an upper plate, (6) is an upper plate gasket, and (7) is a hole through which the rod of the lower plate passes. (8) is a lower plate, (9) is a rod provided in the lower plate (8), (10) is a lower plate gasket, and (11) represents a hole through which the rod of the upper plate passes. 12 is a bolt for tightening the rod 9 of the lower plate and 13 is a bolt for tightening the rod 4 of the upper plate. FIG. 3 is a front view of the combined device to show the perfect sealing effect of the upper and lower and upper and lower plates of a 48-well or 96-well reactor, and FIG. 4 is a body plan view. (14) is a gasket which prevents the outflow of solvent and solute.

FIG. 5 shows a top view and a plan view of the top plate to assist in the sealing role and effective mixing of the flip frame, where (14) represents a rubber plate preventing leakage as mentioned in FIG. 4 above. 14 'represents a disposable gasket that can withstand any solvent by laying on a rubber plate. (15) shows another type of top plate which is different from those mentioned in FIGS. 1 to 4. Numeral 16 shows a form when the upper plate frame is viewed from the front, and FIG. 6 shows a front view and a plan view of the lower plate. In this case (17) is a lower plate, and (17 ') is a view when the lower plate is seen from the front.

7 shows a front view and a plan view after installing the upper plate frame 21 and the lower plate frame 22 in a 48 well or 96 well reactor. At this time (20) is a connecting ring connecting the upper frame and the lower frame. Denoted at 18 is a connection ring connected to the lower frame to fix the lower frame, and 19 at the screw bolt for this connection. 8 is a front view and a plan view after connecting the upper plate 15, the lower plate 17 and all of the upper plate frame 21 and the lower plate frame 22, and tightening the respective connecting screws. As mentioned in Figs. 5 to 7, reference numeral 24 denotes a screw bolt connected to the upper frame.

Existing FlexChem's reactor sealing frame also consists of connecting the upper and lower plates separately, but it is not in the form of a screw to control the degree of sealing during sealing but is sealed in a frame with regular intervals. It is difficult to maintain the state of the initial sealing to the end, and the shielding of the reactor for connection is by hand force, so it is difficult to obtain a perfect sealing effect, and in case of applying excessive stirring force (stirring by rotating power, etc.), Since the contents in the reactor are leaked due to the weakening, the quantitative reaction is not performed. Therefore, a fatal disadvantage is generated in an experiment in which pure compounds are obtained for each well (FIG. 14).

The frame of the present invention has been developed to overcome these disadvantages, the screw type that can be easily screwed to the connecting portion connected to the connecting portion and the degree of close contact between the rubber and the disposable gasket between the reactor and the plate can be adjusted closely Composed of articulations.

When the reaction is carried out using two types of frames, the reactor is completely sealed with the upper and lower frames, and then the reaction is completed. After the final reaction is completed, the compound synthesized in the solid support is separated from the solid support. In order to transfer the dissolved compound to the storage tube, the final lower layer plate is first dismantled. The reason why the top plate is not removed first is that liquid does not flow out to the other open outlet when one outlet is completely sealed. Since the frame of the present invention is completely in contact with each other, even if the bottom plate is removed, the top plate is firmly in contact with each other. Thus, even if the bottom plate is removed, the solution passing through the frit does not leak. In this way, a storage well plate of the same size as the reactor is mounted, and then the top frame is removed.

In order to effectively mix the reactor consisting of a pair of upper and lower frames in the present invention by using a rotating motor with a rotation speed of less than 10 rpm to form a frame that can be equipped with a reactor so that the axis of rotation of the rotating motor is parallel to the ground ( 10) The reactor was mounted parallel to the axis of rotation. That is, the reactor mounted on the sealing frame of the present invention has no fear of leakage, so that the reaction is accelerated while the reaction solution is interlocked from the inlet to the outlet of the well.

In the case of a high temperature reaction according to the reaction conditions it is possible to react by maintaining a temperature above room temperature by installing a rotary motor for mixing in the oven, at a temperature below room temperature to install the reaction apparatus and the mixing device of the present invention in the refrigerator Can be.

In FIG. 10, reference numeral 25 denotes a pedestal on which the reactor is placed, and reference numeral 26 denotes a device capable of adjusting the rotation speed.

In addition, as necessary, by adjusting the length of the reactor mounting frame connected in parallel with the rotating shaft may be equipped with a plurality of reactors.

Compounds synthesized in the reactor have products in the form of mixtures of at least 10 mg and up to 100 mg for compound library construction. The mixture thus synthesized and collected must be purified to the quality of the compound for active screening to be of value as a compound library. Therefore, in the present invention, a compound aggregate synthesized with at least 48 aggregates is introduced into a multiple purification system capable of purifying the aggregate into eight units at a time.

The multiple purification system of the present invention is equipped with eight columns of 10 cm in length and 1.0 to 2.0 cm in diameter to simultaneously purify the compounds in the longitudinal axis wells of the aggregates expected to have similar physical properties (FIG. 11). The desired compound is loaded into each column.

Each column consists of a sample inlet and a solvent inlet, which is a mobile phase, in three directions. When the sample is injected, the solvent inlet is blocked while only the column and the sample inlet are connected. After eight samples are completely injected, the sample inlet is blocked and the solvent inlet is connected to the column so that the solvent passes through the column under the gas pressure of the solvent reservoir. The interchemical interaction between filler and solvent in each column separates the pure compounds in each column and passes through the column. The compound passed in this way may be collected in a tube or tube of 48 wells having a volume of 5.7 ml per well configured to allow eight to be collected in a group. In Fig. 11, reference numeral 27 denotes a sample inlet of the column, 28 denotes a control valve, 29 denotes a column, and 30 denote a fraction outlet.

The solvent reservoir used in the purification system of the present invention uses a transparent container, and consists of a solvent inlet and a gas inlet solvent outlet, and one solvent inlet for dispensing the solvent from the solvent reservoir into eight separate columns. An adapter consisting of eight solvent outlets was used and connected to the adapter of a three-way sample inlet connected between the solvent outlet and the column. In order to separate the mixture in the connected column, the pressure of the gas connected to the solvent reservoir was adjusted to allow an appropriate amount of solvent to flow. Figure 12 shows this system, where (27)-(30) are the same as those mentioned in Figure 11, (31) the fractionation sample tube, (32) the solvent reservoir, and (33) the gas pressure control for the solvent transfer. Device.

In addition, FIG. 13 shows this as a design drawing. (28), (29), (31) and (32) are the same as mentioned in FIG. In this way, the standard set synthesizer and the standard multi-purification system of the present invention can be manufactured inexpensively and easily. It is easy to do this, and moreover, it can be introduced into a purification system and quickly set up a compound library with the quality of the active screening compound and establish a comprehensive system from synthesis to purification by setting up a comprehensive system for combinatorial chemical compound library using solid phase support. System can be said to have been developed. As a result, the genome project resulted in the synthesis of technology related to the construction of a compound library, and thus, a device system that can be used as a standard for constructing a combinatorial chemical compound library using a solid phase support was developed.

Hereinafter, the system of the present invention will be described in more detail with reference to the following Examples, which are intended to illustrate the present invention, but the scope of the present invention is not limited to these Examples.

<Example>

Synthesis of Heidetoin Derivative Library (Figure 1)

48 well aggregate reactors with a volume of 5.0 mL per well are used and 100 mg of Wang resin (1.0 mmol / g) is added per well. Two types of reactors were used. When the connecting ring connected to the sealing bottom plate frame is tightly connected to the bottom plate, the bottom part of the reactor is completely sealed. To load N-α-Fmoc Valine amino acid into Wang resin, 3 mmoles of amino acids, 3 equivalents of wang resin, 3 mmol of dicyclohexylcarbodiimide (DCC), and 0.01 mmol of dimethylaminopyridine (DMAP) Is dissolved in a total of 2.5 mL dimethylformamide (DMF) and added to all 48 wells. In order to proceed with the reaction of the aggregate reactor, the connecting ring connected to the sealing top frame is lifted up and completely fixed to the top plate, and then completely sealed. The sealed reactor was mounted on a mixing rotary mixer frame and reacted at 40 ° C. for 12 hours in an oven. After completion of the reaction, the reactor was disassembled in the rotary mixer frame to remove excess diaminohexylcarbodiimide (DCC), dimethylaminopyridine (DMAP) and dimethylformamide (DMF) solvents. First, the sealing frame of the lower plate was removed, the reactor was placed on the filter device, and then the frame of the upper plate was dismantled. At this time, the intermediate frame connected with the upper and lower frames was washed with resin without disassembly. Resin was washed five times with 2 mL of dichloromethane per well, three times with 2 mL of methanol, three times with 2 mL of dichloromethane, and the resin was dried. In order to remove the Fmoc group, which is an amine protecting group of amino acids linked to Wang resin, a frame was installed in the reactor in the same manner and reacted for 1 hour with 20% piperidine solution to remove the Fmoc protecting group. In order to perform 8 trillion reductive amination reactions with 8 compounds having aldehyde groups, the resin from which Fmoc protecting group was removed was washed and dried several times, and then 8 compounds having aldehyde groups in each well were synthesized. Was dissolved in 1 mL of dimethylformamide (DMF) and added to 1 mL of 10% dimethylformamide (DMF) solution with 3 mmol of sodium triacetoborohydride as a reducing agent per well. Was added. The reaction was reacted at 40 ° C. for 8 hours, the frame was dismantled and the resin washed.

Six isocyanate combinations were added to each well, respectively, and 3 mmol were added to each well, and 2 ml of dimethylformamide (DMF) solvent was added to each well, and reacted at room temperature for 6 hours. Each frame was dismantled, the resin was washed several times and dried, and the final cyclization reaction was carried out for 1 hour under diisopropylamine. After completion of the cyclization reaction, the top plate was brought to the bottom in order to obtain 48 types of hydridein compounds which were separated from the resin and dissolved in diisopropylethylamine (DIEA), and then the bottom plate was dismantled. A plate of the same size that can hold the compound on the reactor in which the lower plate was dismantled was placed on the reactor and turned upside down so that the upper plate faced up. Even in this way, the solvent outflow phenomenon by the sealing frame of this invention was not seen.

The frame of the top plate was removed to receive the compound so that the resin and the compound were not mixed with each other through the reactor filtration frit.

As described above, the compound was purified by the column six times with eighty-eight compounds of 48 compounds (Table 1).

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1 is an elevational view (a) and a cross sectional view (b) of a multiple reactor for a standard assembly,

2 is a sealing substrate and top and bottom plate mounted on the standard reaction multiple reaction vessel,

3 is a front view and a plan view of a hermetic frame equipped with multiple reactors for a standard assembly,

4 is a three-dimensional view of a hermetic frame with multiple reactors for standard assembly,

5 is a front view and a plan view of a top plate of a multiple reactor for another type of standard assembly;

6 is a front view and a plan view of a lower plate of another type of multiple reactor for standard assembly;

7 is a plan view of the intermediate frame of the reactor in 96-well and 48-well form and a plan view of the reaction vessel in which the intermediate frame is fixed on the bottom frame,

8 is a complete view of the reactor in 96-well and 48-well form,

9 is a three-dimensional view of the drawing of FIG. 8;

10 is a rotary mixing device composed of a frame and a rotating motor of the present invention,

11 is a diagram of a standard multiple separation column for building compound diversity of the present invention,

12 is an overall view of purifying a compound on the principle of a flash column,

13 is a schematic drawing of the multi-purification system of the present invention,

14 shows the Flexchem multisynthetic block and sealing system.

Description of each component:

(1) well (1 ') wall plate type reactor

(2) Partitions (3) Outlets

(3 ') frit (4) rod installed on top plate

(5) Tops (6) Tops Gaskets

(7) Hole through which bar of lower plate passes (8) Lower plate

(9) Rods installed on the bottom plate (10) Bottom plate gasket

(11) Holes through which the rod of the top plate passes

(12) (13) Bolts (14) Rubber

(14 ') Disposable Gaskets (15) (16) Tops

(17) (17 ') Bottom plate (18) Hooks connected to bottom frame

(19) Screw bolts connected to the bottom frame

(20) Hooks to fix the top frame

(21) Upper frame (22) Lower frame

(23) Hooks for fixing the top frame

(23 ') Hooks to secure the bottom frame

(24) Screw bolts connected to the top frame

(25) Base (26) Speed Controller

(27) Sample inlet (28) Control valve

(29) column (30) fraction outlet

(31) Sample tubes for fractionation (32) Solvent reservoirs

(33) Gas pressure regulator

.

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Claims (6)

In sealing the well plate reactor 1 ', a rod is attached to the upper plate 5 and the lower plate 8, and the rod 4 provided on the upper plate opens the hole 11 of the lower plate gasket part. The coupling device is fixed by a bolt 13 having a screw shape, and the rod 9 installed on the lower plate passes through the hole 7 of the upper gasket part, and the coupling device is screwed. A through frame for a synthesis reactor configured to completely seal a well plate reactor fixed with a bolt (12). In sealing the well plate reactor 1 ', the lower plate 17, the upper plate frame 21, the lower plate frame 22 and the upper plate 15, and the lower plate, lower plate frame, upper plate frame and The upper plate is connected by flip linkages 23 and 23 'to facilitate connection and detachment of each plate, and the flip linkages 23 and 23' are bolted to fix in a screw form (19 and 24). Flip type frame for the synthesis reactor fixed by. In sealing the well plate reactor 1 ', a rod is attached to the upper plate 5 and the lower plate 8, and the rod 4 provided on the upper plate opens the hole 11 of the lower plate gasket part. The coupling device is fixed by a bolt 13 having a screw shape, and the rod 9 installed on the lower plate passes through the hole 7 of the upper gasket part, and the coupling device is screwed. The rotary mixing device (25, 26) is equipped with a through-frame for the synthetic reaction device that can completely seal the well plate reactor fixed by the bolt (12) by rotating the through-frame by 360 ° Synthesis device for well plate reaction configured. In sealing the well plate reactor 1 ', the lower plate 17, the upper plate frame 21, the lower plate frame 22 and the upper plate 15, and the lower plate, lower plate frame, upper plate frame and The upper plate is connected by flip linkages 23 and 23 'to facilitate connection and detachment of each plate, and the flip linkages 23 and 23' are bolted to fix in a screw form (19 and 24). A well plate reaction synthesis device comprising a rotary mixing device (25, 26) for agitating a flip frame by rotating a flip frame by 360 ° by mounting a flip frame for a synthesis device that is fixed by the same. 5. The synthesizing apparatus as set forth in claim 4, wherein the upper plate (15) and the lower plate (17) have a structure in which a disposable gasket (14 ') resistant to a solvent is laminated on the rubber plate (14) against the outflow rubber plate (14). 5. The sample inlet 27, the control valve 28, the column 29, the fraction outlet 30, and the fraction of the column for separating the reaction product in the synthesis apparatus for the well plate reaction according to claim 3. A well plate reaction synthesis apparatus equipped with a multi-purification column apparatus comprising 8 to 12 columns having a sample tube 31 for use, a solvent reservoir 32, and a gas pressure regulating device for solvent movement 33.